Pesticide Residues and Risk Assessment from Monitoring Programs in the Largest Production Area of Leafy Vegetables in South Korea: a 15-Year Study

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Article Pesticide Residues and Risk Assessment from Monitoring Programs in the Largest Production Area of Leafy Vegetables in South Korea: A 15-Year Study

Duck Woong Park 1,*, Yong Shik Yang 1, Yeong-Un Lee 1, Sue Ji Han 1, Hye Jin Kim 1, Sun-Hee Kim 2, Jong Pil Kim 2, Sun Ju Cho 2, Davin Lee 2, Nanju Song 2, Yujin Han 2, Hyo Hee Kim 2, Bae-Sik Cho 3, Jae Keun Chung 3 and Ae Gyeong Kim 1

1 Gakhwa Agricultural Products Inspection Center, Health and Environment Research Institute of Gwangju, 260, Dongmun-daero, Buk-gu, Gwangju 61138, Korea; [email protected] (Y.S.Y.); [email protected] (Y.-U.L.); [email protected] (S.J.H.); [email protected] (H.J.K.); [email protected] (A.G.K.) 2 Seobu Agro-Fishery Products Inspection Center, Health and Environment Research Institute of Gwangju, 16, Maewol 2-ro, Seo-gu, Gwangju 62072, Korea; [email protected] (S.-H.K.); [email protected] (J.P.K.); [email protected] (S.J.C.); [email protected] (D.L.); [email protected] (N.S.); [email protected] (Y.H.); [email protected] (H.H.K.) 3 Health and Environment Research Institute of Gwangju, 584, Mujin-daero, Seo-gu, Gwangju 61954, Korea; [email protected] (B.-S.C.); [email protected] (J.K.C.) * Correspondence: [email protected] Abstract: Leafy vegetables are widely consumed in South Korea, especially in the form of kimchi Citation: Park, D.W.; Yang, Y.S.; Lee, and namul (seasoned vegetables) and are used for wrapping meat. Therefore, the management of Y.-U.; Han, S.J.; Kim, H.J.; Kim, S.-H.; pesticide residues in leafy vegetables is very important. A total of 17,977 samples (49 leafy vegetables) Kim, J.P.; Cho, S.J.; Lee, D.; Song, N.; were mainly collected in the largest production area of leafy vegetables (Gwangju Metropolitan City et al. Pesticide Residues and Risk and Chonnam Province) in South Korea. They were analyzed within the government’s monitoring Assessment from Monitoring programs (Gwangju Metropolitan City) of pesticide residues between 2005 and 2019. Pesticide Programs in the Largest Production residues were found in 2815 samples (15.7%), and 426 samples (2.4%) from among these exceeded Area of Leafy Vegetables in South the speciﬁed maximum residue limits (MRLs). Samples exceeding the MRLs were mostly detected Korea: A 15-Year Study. Foods 2021, 10, 425. https://doi.org/10.3390/ in spinach, ssamchoo (brassica lee ssp. namai), crown daisy, lettuce, and perilla leaves. Azoxys- foods10020425 trobin, dimethomorph, and procymidone were the most frequently detected pesticides. However, procymidone, diniconazole, and lufenuron were found to most frequently exceed the MRLs. The rate Academic Editor: Mark Mooney of MRLs exceeding has been managed below the average (2.4%) more recently than in the past in this area. Further, leafy vegetables with the most violations of the MRLs in our study in South Korea Received: 20 December 2020 were not harmful to health by a risk assessment (the range of the hazard index was 0.001–7.6%). Accepted: 9 February 2021 Published: 15 February 2021 Keywords: pesticide residue; leafy vegetable; South Korea; azoxystrobin; procymidone; risk assessment

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. A pesticide is any substance or a mixture of chemical substances and is used to protect crops against insects, other pests, fungi, and weeds [1,2]. Further, pesticides are used to modify a plant growth regulator used as a defoliant or desiccant [3]. There are many different kinds of pesticides. Each pesticide is meant to be effective on specific pests. The Copyright: © 2021 by the authors. “-cide” suffix is derived from the Latin word caedere, meaning to kill [4]. The term pesticide Licensee MDPI, Basel, Switzerland. includes all of the following: fungicides, molluscicides, nematicides, insecticides, herbi- This article is an open access article cides, piscicides, avicides, rodenticides, bactericides, insect repellents, animal repellents, distributed under the terms and and antimicrobials [5]. Herbicides are the most common type of pesticide used, accounting conditions of the Creative Commons for almost 80% of all pesticides used by the agriculture sector [6]. Pesticides may remain Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ on or in food after spraying crops. These are known as “pesticide residues” [7]. Many 4.0/). problems arise by eating food (from meat, poultry, and fish to vegetable oils, nuts, and

Foods 2021, 10, 425. https://doi.org/10.3390/foods10020425 https://www.mdpi.com/journal/foods Foods 2021, 10, 425 2 of 17

various fruits and vegetables, etc.) containing pesticide residues. Health effects of pesticide residues may be acute or chronic in those who are exposed depending on the quantity and ways. They may induce negative health effects that have been associated with the immune or nervous system, reproduction, and cancer because pesticides are potentially toxic [8]. Exposure to pesticide residues through food intake can harmfully influence the central nervous system because many pesticides can kill pests by disrupting the nervous system. Organophosphates used mostly in the category of insecticides are especially toxic to the nervous system (inhibitory effects on cholinesterase enzymes) [9]. Despite concerns about their use, pesticides have many advantages, such as the prevention of, reduction in, and elimination of pests at different steps of cultivation and post-harvest in the process of agricultural production. Pesticide usage can help enhance the yield and quality of the produce [10]. Therefore, countries worldwide have been making continuous efforts to ensure proper and safe use of pesticides for many years. In South Korea, the pesticide residue monitoring program began in 1968, and the maximum residue limits (MRLs) regulations for 17 pesticides were first established in South Korea in 1988 [11]. South Korean MRLs were established based on supervised pesticide residue trials. Ensuring safety against residual pesticides in vegetables is especially important in South Korea because vegetables are a very important part of the South Korean diet [12]. Basic Korean dishes such as kimchi (traditionally fermented Korean food) and namul (seasoned vegetables) use plenty of vegetables, especially raw leafy vegetables. These are used to wrap grilled or boiled meat [13]. Pesticides are highly likely to remain in leafy vegetables because leafy vegetables have broad surface areas [14]. Globally, leafy vegetables (raw, boiled, or steamed) consumed account for 2% of total vegetables. Further, these are thought to have a relatively greater health impact than cereal ingestion [14]. Therefore, the management of residual pesticides in the case of leafy vegetables is more important than other types of vegetables. Some leafy vegetables, such as spinach, napa cabbage, and lettuce, are also exported at a mean of roughly 10 million tons/year in South Korea (ATKATI, 2015) [15]. The yields of Gwangju and Chonnam Province account for the largest proportion (38.6%) (Chonnam area accounts for 30.4%) of all types of leafy vegetables produced in South Korea over the past 3 years (KOSTAT 2019) [16]. Therefore, it is necessary to have surveillance programs to analyze the trend of pesticide residues in this area of leafy vegetables. Some studies were conducted on pesticide residues of vegetables for a period of not more than five years in South Korea [13,17–21]. These papers mainly dealt with research on residual pesticides for the entire agricultural product range and health risk assessment. However, this is the first long-term study providing results covering more than a decade from South Korea’s largest leafy vegetable-producing area. Moreover, recently, during a residual pesticide test of leafy vegetables, we found several changes in detection compared to the past. Therefore, we thought it necessary to analyze the change in residual pesticide trends in leafy vegetables by comparing the recent analysis (after 2010) with the results from 10 years ago [13,20,21]. We conducted an analysis of 230 kinds of pesticide residues in 17,977 leafy vegetables (49 kinds) that were imported into Gwangju Metropolitan City (one of the largest consumption cities for leafy vegetables produced in Gwangju Metropolitan City and Chonnam Province) in South Korea over a period of 15 years (from 2005 to 2019). This study is expected to contribute to the management of residual pesticide leafy vegetables not only in South Korea but also on a global scale by analyzing the long-term trends and characteristics of the detection of residual pesticides.

2. Materials and Methods 2.1. Monitoring Programs and Sampling A total of 17,977 samples were collected over 15 years (2005–2019). Sampling was conducted in keeping with the Korea Food Code guideline. The scope of our pesticide residue monitoring program was to analyze 49 types of leafy vegetables according to Foods 2021, 10, 425 3 of 17

Table1. The sampling plan consisted of two main parts. The ﬁrst part involved sampling the incoming wholesale market before the produce was distributed in Gwangju Metropolitan City. The samples analyzed under the government surveillance program (The Health and Environment Research Institute under the Gwangju Metropolitan Government) were collected mainly from the two largest Gwangju area wholesale markets in Gwangju Metropolitan City and Chonnam Province from 2005 to 2019. The second part involved the sampling of leafy vegetables distributed in large or small local markets in Gwangju Metropolitan City. Most of the leafy vegetables consumed and distributed in South Korea were selected, and samples were collected. Whole samples were successfully analyzed within 24 h after collection.

Table 1. The scope of 49 types of leafy vegetables analyzed in our study.

Leafy Vegetable 1. Winter-grown cabbage 16. Mustard leaf 34. Buckwheat leaves 2. Lettuce 17. Kale 35. Dandelion 3. Ssamchoo (brassica lee ssp. 18. Shepherd’s purse 36. Siler divaricata namai) 19. Chicory 37. Broccoli 4. Bomdong (Korean spring 20. Ssam cabbage 38. Amaranth cabbage) 21. Cabbage 39. Brassica campestris 5. Spinach 22. Mugwort narinosa 6. Perilla leaves 23. Chard 40. Sugar beet leaves 7. Crown daisy 24. Butterbur 41. Angelica 8. Marshmallow 25. Narrow-head ragwort 42. Napa cabbage 9. Young radish 26. Chickweed 43. Ponytail radish 10. Aster scaber 27. Fischers ragwort 44. Oak leaf 11. Pepper leaves 28. Red-veined sorrel 45. Red oak lettuce 12. Danggi leaf (Korean 29. Sesame leaves 46. Parsley angelica root leaf) 30. Toscana 47. Pumpkin leaves 13. Pimpinella brachycarpa 31. Wild chive 48. Cabbage lettuce 14. Bok choy 32. Sedum 49. Green mustard leaf 15. Canola (leaf) 33. Arugula

2.2. Reagents and Sample Preparation The Health and Environment Research Institute (Department of Pharmacochemistry) in Gwangju Metropolitan City conducted the tests up until 2010, and from 2011, the tests were conducted by the Agro-Fishery Products Inspection Center at the Health and Envi- ronment Research Institute in Gwangju Metropolitan City. This study used 230 certiﬁed pesticide reference standards purchased from Waco (Osaka, Japan) or Dr. Ehrenstorfer GmbH (Augsburg, Germany) (Table2). Sample preparation was carried out with multiple reaction monitoring (MRM) No. 2 for pesticide residues in keeping with the Korea Food Code. Compounds and solvents (anhydrous sodium, dichloromethane, acetone, n-hexane, and acetonitrile) were obtained from Merck (Darmstadt, Germany). Samples (50 g) ground among the representative portion of the samples (sample size of each vegetable range: 1–2 kg) in a blender (Robot-coupe, South Perkins, Ridgeland, USA) were extracted with 100 ml acetonitrile for 2–3 min. The homogenized mixtures were ﬁltered into a bottle with 10 g anhydrous sodium chloride. The extracts were vigorously vortexed for 1 min. Aliquots of 10 mL were transferred into a tube and evaporated to dryness with a gentle stream of air. Sample extracts for GC (gas chromatographic) analysis were dissolved in 4 ml of 20% acetone/hexane and loaded onto Florisil cartridges (Phe- nomenex, Torrance, CA, USA) (after activating and conditioning). The Florisil cartridges were eluted with 5 ml of 20% acetone/hexane again. The elutions were evaporated with a gentle stream of air and dissolved with 2 ml of acetone for GC analysis. For LC, sample extracts were dissolved with 4 ml of 1% methanol/dichloromethane and loaded onto SPE NH2 Cartridges (Phenomenex, Torrance, CA, USA) (after activating and conditioning). The SPE NH2 Cartridges were eluted with 7 ml of 1% methanol/dichloromethane again. The Foods 2021, 10, 425 4 of 17

elutions were evaporated with a gentle stream of air and dissolved with 2 ml of methanol for LC analysis.

Table 2. Pesticides examined in our study (classiﬁed by pesticide class).

No. Pesticide Class Pesticides 1 1,3,5-triazine terbuthylazine 2 2,6-dinitroaniline fungicide fluazinam 3 Alkanamide (acetamide) diphenamid 4 Anilide herbicides; phenoxy herbicides clomeprop 5 Anilinopyrimidine cyprodinil, mepanipyrim, pyrimethanil Aromatic hydrocarbon; 6 dicloran, quintozene, tolclofos-methyl chlorophenyl/nitroaniline 7 Arylpyrrole chlorfenapyr 8 Benzenesulfonamide flusulfamide 9 Benzamide fungicide zoxamide 10 Benzilate bromopropylate triflumuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, 11 Benzoylurea teflubenzuron 12 Benzoisothiazole probenazole fenobucarb, furathiocarb, pirimicarb, aldicarb, bendiocarb, 13 Carbamate carbaryl, carbofuran, ethiofencarb, isoprocarb, methiocarb, methomyl, oxamyl, propoxur 14 Carboxamide flutolanil, thifluzamide, mepronil, boscalid 15 Chloroacetamide propisochlor, dimethenamid, thenylchlor 16 Chloronitrile chlorothalonil 17 Cinnamic acid amide dimethomorph 18 Cyclodiene organochlorine BHC, endosulfan, chlordane 19 Diacylhydrazine chromafenozide, methoxyfenozide, tebufenozide 20 Dicarboximide iprodione, procymidone, vinclozolin 21 Dinitroaniline pendimethalin 22 Dithiolane isoprothiolane 23 Fungicides (aryl phenyl ketone fungicides) metrafenone Fungicides(sulfonamide fungicides; triazole 24 amisulbrom fungicides) 25 Hydroxyanilide fenhexamid 26 Imidazole imazalil, prochloraz, triflumizole 27 Isobenzofuranone fthalide 28 Juvenile hormone mimic fenoxycarb, pyriproxyfen 29 Methoxyacrylate azoxystrobin 30 Methoxycarbamate pyraclostrobin 31 Mite growth inhibitor etoxazole 32 Neonicotinoid acetamiprid, clothianidin, thiacloprid, thiamethoxam 33 N-phenyl carbamate fungicide diethofencarb 34 N-phenylphthalimide flumioxazin Foods 2021, 10, 425 5 of 17

Table 2. Cont.

No. Pesticide Class Pesticides azinphos-methyl, EPN, ethion, mecarbam, methidathion, parathion-methyl, phenthoate, phosmet, phosphamidone, cadusafos, chlorpyrifos, chlorpyrifos-methyl, diazinon, 35 Organophosphate dichlorvos, dimethoate, dimethylvinphos, ethoprophos, fenitrothion, fenthion, fosthiazate, malathion, parathion, phosalone, pirimiphos-methyl, profenofos, prothiofos, pyraclofos, quinalphos, tebupirimfos, terbufos, thiometon, triazophos 36 Phenylamide: butyrolactone ofurace 37 Phenylamide: oxazolidinone oxadixyl 38 Phenylpyrazole fipronil 39 Oxadiazine indoxacarb 40 Oxazolidinedione pentoxazone 41 Oxime carbamate butocarboxim, thiodicarb 42 Oximinoacetate trifloxystrobin 43 Oxyacetamide mefenacet, flufenacet 44 Phenylpyrrole fludioxonil 45 Phthalimide captafol, captan, folpet 46 Phosphorothiolate edifenphos, iprobenfos, pyrazophos 47 Propionamide fenoxanil acrinathrin, bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, 48 Pyrethroid deltamethrin, fenpropathrin, fenvalerate, permethrin, tefluthrin, tralomethrin 49 Pyridine dithiopyr, thiazopyr 50 Pyrimidine fenarimol, nuarimol 51 Pyrimidinol bupirimate 52 Pyrimidinyloxybenzoic pyriminobac-methyl 53 Pyrroloquinolinone pyroquilon 54 Pyrazole herbicide pyrazolate 55 Qil cyazofamid 56 Strobilurin type- methoxyacrylate picoxystrobin 57 Strobilurin type: oximinoacetate kresoxim-methyl 58 Sulfamide dichlofluanid 59 Sulfonylurea cinosulfuron 60 Tetronic acid spirodiclofen 61 Thiocarbamate Esprocarb, molinate, pyributicarb 62 Thiazole carboxamide ethaboxam 63 Thiazdiazole carboxamide tiadinil 64 Triazolobenzothiazole tricyclazole diniconazole, fenbuconazole, penconazole, triadimefon, 65 Triazole uniconazole, cyproconazole, flusilazole, metconazole, myclobutanil, simeconazole, fluquinconazole, imibenconazole Foods 2021, 10, 425 6 of 17

Table 2. Cont.

No. Pesticide Class Pesticides 66 Uracil bromacil 67 Valinamide carbamate iprovalicarb 68 Urea methabenzthiazuron aldrin, binapacryl, butafenacil, carbophenothion, chinomethionat, chlorobenzilate, cyanazine, cyflufenamid, DDT, dicofol, dieldrin, diflufenican, dimethachlor, endrin, flonicamid, flumiclorac pentyl, fluthiacet-methyl, fluvalinate, heptachlor, indanofan, lactofen, mefenpyr-diethyl, methoxychlor, nitrapyrin, nitrothal-isopropyl, pyridalyl, pyrimidifen, tetradifon, ametryn, anilofos, azaconazole, cyanophos, dimepiperate, 69 The others diphenylamine, etrimfos, fenazaquin, fenothiocarb, tolyfluanid, fonofos, isazofos, isofenphos, isoxathion, paclobutrazol, pirimiphos-ethyl, propazine, pyridaben, tebufenpyrad, triticonazole, 3,4,5-Trimethacarb, benzoximate, chlorantraniliprole, cymoxanil, fenpyroximate, ferimzone, fluacrypyrim, forchlorfenuron, metolcarb, oxaziclomefon, promecarb, pyribenzoxim, quinoclamine

2.3. Instrumental Analysis Instrumental analysis was performed using GC (gas chromatography) instruments and apparatuses and LC (liquid chromatography) instruments and apparatuses. The details were as follows: the GC-nitrogen phosphorous detector (NPD) system (for organophosphorus and nitrogen-containing compounds) and the GC-63Ni electron capture detector (ECD) system (for organochlorine and pyrethroid compounds) were used for GC analysis. An Agilent 7890 series GC instrument coupled to the NPD and ECD was used for analysis. The chromatographic separation was accomplished using a DB-5 column (Agilent, Santa Clara, CA, USA). The confirmation of residues was carried out with an Agilent 6890 series GC combined with an HP 5973 MSD (mass selective detector). A DB-5 (0.25 mm × 30 m, 0.25 µm film thickness; Agilent) column for GC-NPD and GC-ECD, and a DB-5MS (0.25 mm × 30 m, 0.25 µm film thickness; Agilent) column for GC-MS (mass spectrometry) were used for separation. The GC analysis conditions were as follows: 1. GC-NPD: The column temperature was programmed from 190 to 240 ◦C (held for 3 min) with an increase of 4◦C/min and increased to 290◦C at 20◦C/min (held for 5 min). 2. GC-ECD: The column temperature was programmed from 190 to 220 ◦C (held for 10 min) with an increase of 12◦C/min and increased to 290◦C at 7◦C/min (held for 6 min). 3. GC-MS: The column temperature was programmed from 190 to 290 ◦C (held for 5 min) with an increase of 10◦C/min. LC analysis was performed using a Waters UPLC (Ultra Performance Liquid Chro- matography) Acquity H-class (Waters Corporation, Milford, MA, USA) and a TSQ Quan- tum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, Miami, FL, USA). A reversed-phase Acquity UPLC BEH C18 LC column (2.1 × 50 mm, 1.7 µm; Acquity Group, Chicago, IL, USA) was used for chromatographic separation in UPLC. The TSQ Quantum Ultra triple quadrupole mass spectrometer and the UPLC were used in com- bination. Electrospray ionization was performed in positive and negative modes. Data were obtained in MRM mode. The capillary temperature of the MS was 300 ◦C, and the ion spray voltage was 3500 eV. Collision-induced dissociation was conducted with argon. Collision cell argon gas pressure was set to 1.5 mTorr. Chromatographic analyses were performed with gradient elutions (eluent A: formic acid (0.1%) and methanol–water (98:2, v/v), and eluent B: formic acid (0.1%) and methanol). Gradient elutions started at 95% of eluent A and 5% of eluent B for 0.2 min. This step was held for an additional 3.0 min before being returned to 100% of eluent B. The overall running time was 6 min. The separation of Foods 2021, 10, 425 7 of 17

analytes was performed with a ﬂow rate of 0.45 mL/min. The column temperature was maintained at 40 ◦C. The injection volume was 1 µL.

2.4. Method Validation Analysis of the lettuce matrix was used for validation of the sample preparation and analytical methods, linearity, LOD, LOQ, and recovery. The analytical methods validation was conducted according to the SANTE guidelines [22]. Since our study was a long- term study for 15 years, validation tests were carried out three times during the research period. This was conducted simultaneously with other previous studies conducted in our institution [13,20,21]. We used Excel 2016 (Microsoft, Redmond, WA, USA) to calculate the average value of residual pesticide detection and nonconformity.

3. Results and Discussion 3.1. Method Validation A total of 19 different pesticides detected at more than 1% (pesticides MRLs) were chosen for the method validation. The validation results met the validation parameters and criteria (sensitivity, linearity, recovery, precision, and accuracy) in the SANTE guidelines [22]. The sensitivity of the method was conﬁrmed with the LOD and LOQ. An S/N of 3:1 and 10:1, respectively, was accepted. The matrix-matched calibrations were obtained for the linearity test by plotting the area of each target pesticide against the concentrations of the calibration standards. Mean recoveries from the initial validation should be within the range 70–120%, with an associated repeatability RSD ≤ 20% for precision, for all analytes within the scope of a method, according to the SANTE guidelines [22]. The results shown in Table3 met this criterion. Table3 represents the results of the validation testing. The correlation coefﬁcient showed good linearity of 0.9902-0.9999. Percent recoveries were 86.2–109.5% for all pesticides. The standard deviation of the recovery rate was < 5.7%. The LOD of pesticides analyzed were from 0.003 to 0.043 µg g–1 and LOQ ranged between 0.010 and 0.129 µg g–1. Results from this study indicate that the analytical methods were appropriate for the analysis of pesticide residues in our study.

Table 3. Validation parameters (linearity, LOD, LOQ, and recoveries) in main pesticides detected in our study.

Correlation Recovery ± Pesticide LOD (µg g–1) a LOQ (µg g–1) b Coefﬁcient (r2) RSD (%) Azoxystrobin 0.9991 0.041 0.124 90.1 ± 3.4 Boscalid 0.9998 0.020 0.059 94.1 ± 3.4 Chlorfenapyr 0.9978 0.038 0.033 105.5 ± 1.0 Chlorothalonil 0.9988 0.023 0.071 93.9 ± 1.7 Chlorpyrifos 0.9995 0.007 0.020 95.5 ± 3.2 Cyazofamid 0.9990 0.012 0.035 92 ± 2.1 Diazinon 0.9998 0.003 0.011 86.2 ± 3.8 Dimethomorph 0.9975 0.033 0.100 101 ± 1.9 Diniconazole 0.9992 0.008 0.026 99.5 ± 0.4 Endosulfan 0.9997 0.005 0.015 88.7 ± 0.6 Ethaboxam 0.9999 0.003 0.010 97 ± 1.2 Ethoprophos 0.9996 0.006 0.017 89.9 ± 3.9 Fenitrothion 0.9997 0.005 0.016 109.5 ± 2.7 Flufenoxuron 0.9992 0.043 0.129 100.0 ± 5.7 Indoxacarb 0.9994 0.007 0.021 102.0 ± 0.6 Lufenuron 0.9967 0.016 0.049 100.4 ± 2.7 Procymidone 0.9997 0.005 0.014 87.1 ± 1.6 Pyraclostrobin 0.9902 0.036 0.110 93 ± 5.5 Pyridalyl 0.9987 0.011 0.032 101.3 ± 4.1 a: limit of detection, b: limit of quantiﬁcation. Foods 2021, 10, 425 8 of 17

Foods 2020, 9, x FOR PEER REVIEW 20 of 19 3.2. Trends in Annual Pesticide Residue Levels were Thisanalyzed is the for ﬁrst pesticide long-term residues study from of pesticide 2005 to 2019 residues (Figure in leafy 1). A vegetablestotal of 4.773 conducted samples werein areas analyzed with the by highestthe Health production and Environmen volumest in Research South Korea. Institute A total (Department of 17.977 of samples Phar- macochemistry)were analyzed for (2005–2010), pesticide residues and 13.204 from 2005samp toles 2019 were (Figure analyzed1). A totalby the of 4.773Agro-Fishery samples Productswere analyzed Inspection by the Center Health established and Environment by the Health Research and Institute Environment (Department Research of Institute Pharma- Figurecochemistry) 1. Samples (2005–2010), analyzed and by 13.204 the monitoring samples were programs analyzed of bypesticide the Agro-Fishery residues (Gwangju Products MetropolitanInspection Center City) established from 2005 byto 2019: the Health Trends and in Environmentthe annual number Research and Institute percent Figure (pesti-1. cides(2011–2019). residues < MRLs and > MRLs). (2011–2019).

Figure 1. SamplesSamples analyzed analyzed by by the the monitoring monitoring programs programs of of pesticide pesticide residues residues (Gwangju (Gwangju Metropolitan Metropolitan City City)) from from 2005 to 2019: Trends in the annual number and percent (pesticides residues < MRLs and > MRLs). 2019: Trends in the annual number and percent (pesticides residues < MRLs and > MRLs).

PriorPrior to thethe establishmentestablishment of of the the inspection inspection center, center, less less than than 1000 1000 tests tests were were conducted con- ductedannually, annually, between between 544 and 544 945. and In 2011,945. In an 2011, agricultural an agricultural inspection inspection agency wasagency established, was established,and since and then, since the numberthen, the of number annual inspectionsof annual inspections has doubled. has doubled. TheThe number ofof samples samples with with pesticide pesticide residues residues below below the MRLsthe MRLs is 2.389 is (13.3%)2.389 (13.3%) among amongthe 17,977 the leafy17,977 vegetable leafy vegetable samples samples over the 15-yearover the period. 15-year In period. addition, In samplesaddition, exceeding samples exceedingthe MRLs totalthe MRLs 426 (2.4%). total 426 From (2.4%). 2005 From to 2007, 2005 residues to 2007, below residues the MRLs below tended the MRLs to be tended higher tothan be thehigher average than (Figurethe average1.). We (Figure can see 1.). that We thecan nonconformity see that the nonconformity rate has decreased rate has since de- creased2005, and since in particular, 2005, and itin hasparticular, been below it has average been below since average 2011. The since number 2011. of The samples number with of samplespesticide with residues pesticide above residues MRLs wasabove also MRLs higher wa thans also the higher average than (2.4%) the average from 2005(2.4%) to from 2007. 2005Overall, to 2007. there Overall, was a high there percentage was a high of percen samplestage with of samples pesticide with residues pesticide below residues the MRLs be- lowand the above MRLs the and MRLs, above between the MRLs, 2005 between and 2007. 2005 It and can 2007. be seen It can that be the seen pattern that the of pattern sample ofproportions sample proportions with pesticide with residuespesticide below residues the MRLsbelow andthe MRLs above and the MRLsabove isthe not MRLs consistent. is not consistent.Sample portions Sample with portions pesticide with residues pesticide below residues the MRLs below were the detectedMRLs were at a higherdetected rate at ina higher2014–2018 rate thanin 2014–2018 in 2008–2013. than However,in 2008–2013. the proportion However, ofthe nonconformities proportion of nonconformities remained below remainedaverage between below average 2014 and between 2018 (Figure2014 and1). 2018 We think(Figure farmers 1.). We usedthink pesticides farmers used correctly pesti- cidesaccording correctly to the according method ofto use.the method The rate of of us nonconformitye. The rate of nonconformity was the highest was in 2005, the highest but it is indecreasing, 2005, but it and is decreasing, it seems that and it it has seems been that managed it has been below managed the nonconformity below the nonconform- average for itythe average last nine for years. the last The nine causes years. of theThe management causes of the belowmanagement the nonconformity below the nonconform- average are itythought average to beare as thought follows. to be as follows. When nonconformities are detected, the following procedures are carried out in South Korea: The Agricultural Products Inspection Center conducts residual pesticide tests on agricultural products entering the market and agricultural products distributed in the region. When nonconformities are found in the tests, administrative actions are

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When nonconformities are detected, the following procedures are carried out in South Korea: The Agricultural Products Inspection Center conducts residual pesticide tests on agricultural products entering the market and agricultural products distributed in the region. When nonconformities are found in the tests, administrative actions are taken through the relevant county ofﬁces and district ofﬁces. Restrictions are imposed on farms that violate the law (treatment fees and suspension of shipments). The National Agricultural Products Quality Management Service in South Korea visits the farm and inspects the produce again. These efforts are combined to enable farmers to use pesticides accurately in South Korea.

3.3. Evaluation of Pesticide Residue Levels by Pesticide Type In 2.389 samples, we detected pesticide levels below the MRLs. Table4 summarizes the main pesticides detected in leafy vegetables. Azoxystrobin (17.8%, 425/2.389), dimethomorph (16.5%, 393/2.389), procymidone (11.3%, 271/2.389), indoxacarb (6.7%, 160/2.389), and lufenuron (5.1%, 122/2.389) were the most frequently detected pesticides (with low MRLs). These types of pesticides account for nearly 58% of the total amount. However, Figure2 shows that these values changed over the long term. The most frequently detected azoxystrobin was rarely detected before 2010. The detection increased after 2011, and it was frequently detected until 2018. Azoxystrobin is a broad-spectrum systemic fungicide of the class of methoxyacrylates (ARS 2008) [23]. Some pesticides, such as procymidone and endosulfan, were frequently detected, but our results show that their concentrations have decreased in recent years. Pesticides such as procymidone, widely used in South Korea, are managed through farm management by concerned authorities (The National Agricultural Products Quality Management Service, county offices, and district offices). Therefore, it seems that the frequency of detection of procymidone has decreased compared with that in the past. Endosulfan is a harmful agrochemical as an organochlorine insecticide and acaricide due to its acute toxicity and potential for bioaccumulation. Further, it is considered as a potential endocrine disruptor [24]. A worldwide ban on the production and use of endosulfan was agreed under the Stockholm Convention in April 2011 [25]. Since 2011, endosulfan has been banned from being manufactured and used in South Korea. The management system has been strengthened since 2015, as it was included in the Korean persistent organic pollutants (POPs) [26]. As shown in Figure2, the use of endosulfan decreased after 2011. Despite the ban on the use of endosulfan, one detection per year in 2011 and 2014–2016 was made based on our research. Based on these results, these pesticides need to be managed continuously via official monitoring programs in case they are used. Indoxacarb was detected before 2014, from 2005 to 2010. It was not detected from 2011 to 2014 and has been detected again since 2015 (4–36 times per year). Pyridalyl was detected for the first time in our tests in 2017, and its detection (6–26 times per year) has increased in the last three years (2017–2019). The order of the high detection of pesticide residues (number of pesticides ions, lambda-cyhalothrin, deltamethrin, and dithiocarbamates were found [30]. These results show that the pesticide components, which are mainly used in leafy vegetables, vary from country to country. Foods 2021, 10, 425 10 of 17

Table 4. Pesticides most frequently detected (number of pesticides MRLs (%)) in the samples analyzed in our study from 2005 to 2019.

Number of Pesticides < MRLs (%) Number of Pesticides > MRLs (%) Pesticide Pesticide (Total: 2.389) (Total: 426) Azoxystrobin 425 (17.8%) Procymidone 40 (9.4%) Dimethomorph 393 (16.5%) Diniconazole 37 (8.7%) Procymidone 271 (11.3%) Lufenuron 33 (7.8%) Indoxacarb 160 (6.7%) Diazinon 32 (7.5%) Lufenuron 122 (5.1%) Chlorpyrifos 30 (7.0%) Endosulfan 112 (4.7%) Endosulfan 28 (6.6%) Diniconazole 88 (3.7%) Azoxystrobin 26 (6.1%) Boscalid 87 (3.6%) Indoxacarb 22 (5.2%) Chlorothalonil 74 (3.1%) Dimethomorph 16 (3.8%) Flufenoxuron 60 (2.5%) Chlorfenapyr 15 (3.5%) Ethaboxam 50 (2.1%) Chlorothalonil 11 (2.6%) Diazinon 49 (2.1%) Boscalid 9 (2.1%) Pyridalyl 47 (2.0%) Flufenoxuron 8 (1.9%) Pyraclostrobin 46 (1.9%) Ethoprophos 7 (1.6%) Cyazofamid 39 (1.6%) Fenitrothion 7 (1.6%) The others 366 (15.3%) The others 105 (24.6%)

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Azoxystrobin Dimethomorph Procymidone Indoxacarb Lufenuron Endosulfan Diniconazol Boscalid Chlorothalonil Flufenoxuron Ethaboxam Diazinon Pyridalyl Pyraclostrobin Cyazofamid

Figure 2. Distribution of pesticide residues (

samples analyzed. 3.4. Violations of MRLs and Health Risk Assessment In this study, 49 types of leafy vegetables (17,977 samples) were analyzed mainly for 230 pesticides. The number of pesticides over the MRLs in main leafy vegetables is described in Table 5.

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3.4. Violations of MRLs and Health Risk Assessment In this study, 49 types of leafy vegetables (17,977 samples) were analyzed mainly for 230 pesticides. The number of pesticides over the MRLs in main leafy vegetables is described in Table5.

Table 5. Main pesticides detected in the 15 highest leafy vegetables exceeded MRLs in our study from 2005 to 2019.

Number of Number of Main Pesticides Range of Pesticides Samples Pesticides (%) Mrls Vegetables Pesticides Exceeded Mrls Found in Samples Analyzed < MRL And (Mg Kg–1) > MRL (%) (No.) (Mg Kg–1) > MRL Chlorpyrifos (9) 0.01 → 0.05 * 0.06 ~ 0.92 Spinach 1.283 354 (27.6) 49 (3.8) Lufenuron (9) 0.2 → 5.0 * 0.4 ~ 3.8 Indoxacarb (6) 1.0 → 3.0 * 1.1 ~ 7.2 Chlorothalonil(4) 5.0 5.5 ~ 22.1 Ssamchoo(brassica Diniconazole (19) 0.3 0.4 ~ 6.6 119 (26.2) 38 (8.4) lee ssp. Namai) 455 Diazinon (11) 0.1 0.2 ~ 3.4 Diazinon (7) 0.1 → 0.01 * 0.09 ~ 1.79 Crown daisy 940 145 (15.4) 36 (3.8) Lufenuron (4) 0.2 → 5.0 * 0.3 ~ 2.1 Procymidone (3) 5.0 → 0.05 * 8.40 ~ 11.40 Endosulfan (4) 1.0 → 0.05 * 0.30 ~ 6.20 Lettuce 2.632 212 (8.1) 34 (1.3) Procymidone (4) 5.0 6.3 ~ 10.7 Chlorpyrifos (3) 0.01 0.07 ~ 0.30 Lufenuron (3) 0.2 → 7.0 * 0.4 ~ 2.1 Diniconazole (9) 0.3 0.7 ~ 2.4 Perilla leaves 1.369 324 (23.7) 32 (2.3) Azoxystrobin (4) 2.0 → 20 * 4.4 ~ 20.7 Pimpinella 459 132 (28.8) 25 (5.4) Procymidone (15) 5.0 → 0.05 * 0.17 ~ 35.68 brachycarpa Procymidone (5) 5.0 → 0.05 * 0.31 ~ 34.4 Azoxystrobin (2) 3.0 4.0, 9.4 Aster scaber 456 157 (34.4) 21 (4.6) Chlorpyrifos (2) 0.01 4.77, 0.34 Diazinon (2) 0.1 → 0.01 * 1.3, 3.9 Parathion (2) 0.3 → 0.01 * 0.60, 1.20 Endosulfan (3) 0.1 → 0.05 * 0.20 ~ 2.00 Chicory 779 71 (9.1) 18 (2.3) Procymidone (3) 5.0 → 0.05 * 3.22 ~ 9.50 Danggi leaf Azoxystrobin (4) 2.0 → 20 * 3.0 ~ 12.3 (Korean angelica 41 (26.5) 17 (11.0) 155 → root leaf) Chlorfenapyr (3) 0.5 0.7 * 1.3 ~ 1.8 Procymidone (3) 5.0 → 0.05 * 0.41 ~ 12.50 Chlorpyrifos (3) 0.01 → 0.15 * 0.22 ~ 1.47 Mustard green 168 47 (28.0) 16 (9.5) EPN (2) 0.1 → 0.01 * 5.30, 6.50 Lufenuron (2) 0.2 → 5.0 * 0.6, 0.8 Endosulfan (3) 1.0 → 0.05 * 0.30 ~ 1.8 Marsh mallow 451 62 (13.7) 16 (3.5) Triﬂoxystrobin (3) 0.5 → 20 * 1.1 ~ 1.5 Endosulfan (3) 1.0 → 0.05 * 1.00 ~ 4.70 Winter-grown 218 (24.0) 16 (1.8) cabbage 909 Diazinon (2) 0.1 0.3, 0.7 EPN (2) 0.2 → 0.01 * 4.1, 6.0 Procymidone (2) 5.0 → 0.05 * 7.60, 15.80 Acrinathrin (3) 0.1 → 5.0 * 0.5 ~ 1.5 Pepper leaves 96 37 (38.5) 13 (13.5) Boscalid (3) 0.3 → 0.01 * 0.28 ~ 17.40 Diazinon (3) 0.05 → 0.01 * 0.48 ~ 0.68 Young radish 678 164 (24.2) 13 (1.9) Procymidone (2) 0.05 0.20, 0.51 EPN (2) 0.05 → 0.01 * 7.10, 12.97 Boscalid (2) 0.3 0.9, 3.0 Diazinon (2) 0.05 → 0.01 * 0.31, 3.14 Sesame leaves 312 70 (22.4) 10 (3.2) Diniconazole (2) 0.3 0.5, 0.6 Note: * Korean MRLs that have changed during our study period (2005–2019). Foods 2021, 10, 425 12 of 17

A total of 61 pesticides (436 times) were detected in 426 samples that exceeded the MRLs. According to the food safety management guidelines, the top items above the MRLs in leafy vegetables in research from all over South Korea were as follows: perilla leaves, spinach, lettuce, napa cabbage, crown daisy, pepper leaves, pimpinella brachycarpa, chard [31–33]. These findings are generally consistent with the results of our study. How- ever, there was one characteristic difference. In our results, ssamchoo (brassica lee ssp. namai) is the second highest in the leafy vegetables exceeding the MRLs. It is thought to be the reason why there were more inflows compared to other cities as a consumption site near the production area of ssamchoo (brassica lee ssp. namai). In studies of other countries around the world, the pesticides most frequently found above the MRLs in cabbage were mainly procymidone, in Brazilian monitoring programs [1]. Metalaxyl, boscalid, chlorothalonil, and difenoconazole exceeded the MRLs for chard, and mancozeb, difenoconazole, lambda-cyhalothrin, and thiamethoxam exceeded the MRLs for lettuce in Chile [28]. Additionally, the pesticides most frequently found above the MRLs in pak choi were chlorpyrifos in China [27]. We could see that procymidone, boscalid, chlorothalonil, and chlorpyrifos were the main pesticides, similar to our results, that caused nonconformities in leafy vegetables in other countries. Compared with Figure3, the overall results of the country are similar to those of our study results between 2007 and 2012, with the highest detection of endosulfan as a whole, and procymidone, chlorpyrifos, diazinon, azoxystrobin, and diniconazole are also high. However, lufenuron was the highest detected pesticide in 2005 and 2009, with minor differences (Figure3). In the long term, we can see that the trend of pesticides violating the MRLs changes, as shown in Figure3. From 2005 to 2009, lufenuron, azoxystrobin, and diazinon were the top pesticides that violated the MRLs. Characteristically, from 2010, the proportion of lufenuron in nonconformities decreased. Further, diniconazole tended to account for a high proportion. As a result, there has been a change in the trend of pesticides violating the MRLs. Table5 shows that the MRLs rate of certain agricultural chemicals is high for the following agricultural produce: chlorpyrifos, lufenuron in spinach, diniconazole in ssamchoo (brassica lee ssp. namai), procymidone in pimpinella brachycarpa, and aster scaber. These results indicate that certain leafy vegetables have been identified with violations of the MRLs for specific pesticides. Based on these results, leafy vegetables that were frequently detected with certain pesticides need to be intensively managed. Even with the same pesticide, various MRLs are set up for each agricultural product, and it seems to have been used without accurate information on agricultural products with exceptionally low MRLs. Ssamchoo (brassica lee ssp. namai) was developed in South Korea in 1998 and created through the interspecies hybridization of cabbages and napa cabbages. It is a new vegetable that has completely different ecological characteristics, shapes, and genetic composition than those of cabbage and Korean cabbage varieties [34]. In South Korean food culture, people wrap meat and rice in leafy vegetables. Therefore, if ssamchoo (brassica lee ssp. namai) grows too large, it is less commercialized. It is thought to use many pesticides, such as diniconazole, to control growth to a moderate size. Diniconazole is a broad-spectrum triazole fungicide that acts as a plant growth regulator, decreasing the height and leaf area in bean plants when applied to roots [35,36]. Figure4 shows the trend of pesticides over the MRLs for each agricultural product. This shows the level of pesticide residue management for each vegetable in the long run. For example, lettuce, spinach, and perilla leaves, the representative leafy vegetables consumed in South Korea, seem to be systematically managed because of the low number of violations of MRLs compared with that in the past. On the other hand, MRLs violations occur sporadically in crown daisy, aster scaber, danggi leaf (Korean angelica root leaf), marshmallow, and pepper leaves. Therefore, it is necessary to continuously check various vegetables for residual pesticides in order to prevent safety blind spots on agricultural products. Foods 2021, 10, 425 13 of 17

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Azoxystrobin Boscalid Chlorfenapyr Chlorothalonil Chlorpyrifos Dimethomorph Endosulfan Epn Flufenoxuron Indoxacarb Lufenuron Procymidone Diniconazol Pyraclostrobin Diazinon Ethoprophos Fenitrothion Fludioxonil Thiacloprid Thiamethoxam Fenpyroximate Fluquinconazole Kresoxim-methyl Trifloxystrobin Metconazole Carbofuran Acrinathrin Pyridalyl

Figure 3. Annual distribution of pesticide residues exceeding the maximum residue limits (MRLs) in samples analyzed Figure 3. Annual distribution of pesticide residues exceeding the maximum residue limits (MRLs) in samples analyzed from 2005 to 2019. from 2005 to 2019. However, lufenuron was the highest detected pesticide in 2005 and 2009, with minor differences (Figure 3). In the long term, we can see that the trend of pesticides violating the MRLs changes, as shown in Figure 3. From 2005 to 2009, lufenuron, azoxystrobin, and diazinon were the top pesticides that violated the MRLs. Characteristically, from 2010, the proportion of lufenuron in nonconformities decreased. Further, diniconazole tended to

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Foods 2020, 9, x FOR PEER REVIEW 28 of 19

Figure 4. Annual distribution by which the MRLs are exceeded in the main leafy vegetables analyzed from 2005 to 2019 Figure 4. Annual distribution by which the MRLs are exceeded in the main leafy vegetables analyzed from 2005 to 2019 (blue lines—the nonconformity rate of samples, orange bars—the number of nonconformities of samples). (blue lines—the nonconformity rate of samples, orange bars—the number of nonconformities of samples).

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Based on our results, we conducted the exposure evaluation with pesticides that exceeded the MRLs. We chose four kinds of leafy vegetables (spinach, ssamchoo (brassica lee ssp. Namai), lettuce, perilla leaves). These vegetables with the most violations of MRLs in our study are consumed a lot in South Korea. A risk assessment was conducted with the estimated daily intake (EDI) and acceptable daily intake (ADI). The ADI is an estimate of the maximum amount of a substance consumed daily over a lifetime without an appreciable health risk [37]. The calculation of the EDI was made using the pesticide residue results and average food consumption per person per day [31]. The calculated results are demonstrated in Table6.

Table 6. Risk assessment of pesticides violating the MRLs in 5 main leafy vegetables.

Average EDI (mg/ ADI (mg/ Hazard Vegetables Pesticide Concentration person/day) (a) person/day) (b) Index(%) (c) (mg kg–1) Chlorpyrifos 0.32 0.00192 0.55 0.349091 Lufenuron 1.47 0.00882 0.77 1.145455 Spinach Indoxacarb 3.67 0.02202 0.55 4.003636 Chlorothalonil 14.02 0.08412 1.1 7.647273 Ssamchoo (brassica Diniconazole 1.38 0.00001 1.1 0.001255 lee ssp. Namai) Diazinon 1.27 0.00001 0.275 0.004618 Diazinon 1.27 0.00064 0.275 0.230909 Crown daisy Lufenuron 1.45 0.00073 0.77 0.094156 Procymidone 9.13 0.00457 5.5 0.083 Endosulfan 2.22 0.01288 0.33 3.901818 Procymidone 8.3 0.04814 5.5 0.875273 Lettuce Chlorpyrifos 0.22 0.00128 0.55 0.232 Lufenuron 1.06 0.00615 0.77 0.798442 Diniconazole 1.17 0.00304 1.1 0.276545 Perilla leaves Azoxystrobin 14.87 0.03866 11.0 0.351473 (a) Average concentration (mg/kg) × Daily food intake (g/person/day)/1000. (b) Acceptable daily intake (mg/kgbw/day) × 55 kg. (c) Hazard index (%ADI) = (EDI/ADI) × 100.

The range of EDIs calculated was 1.0 × 10–5−8.4 × 10–2. The range of the hazard index was 0.001–7.6%. The ratio for chlorothalonil in spinach was 7.6% and the highest in the risk assessment test. A hazard index (percent of EDI to ADI ratios) over 100% indicates the possibility that the exposure would induce obvious toxic effects [38]. Further, leafy vegetables with the most violations of MRLs in our study in South Korea were not harmful to health by a risk assessment. However, as chronic exposure to pesticides may reduce cognitive abilities, the elderly and the weak should be careful about the intake and exposure of pesticides [39].

4. Conclusions Among 17,977 samples, pesticides were detected in 2.815 samples (15.7%), of which 426 samples (2.4%) exceeded the MRLs. In our study, lettuce and perilla leaves, the representative leafy vegetables consumed in South Korea, seem to be managed due to the low number of violations of MRLs compared with that in the past. In our study, certain leafy vegetables were identiﬁed with the violation of MRLs for speciﬁc pesticides, and it seems that special care is needed. Moreover, leafy vegetables with the most violations of MRLs in our study in South Korea were not harmful to health by a risk assessment (the range of the hazard index was 0.001–7.6%). There were no results such as a long-term residual pesticide detection change as in our paper for leafy vegetables worldwide. Therefore, we think our results are a good prior study for the long-term study of residual pesticides in other countries. Further, because some leafy vegetables (spinach, napa cabbage, lettuce, etc.) in South Korea are exported abroad, our results are expected to be used as good data for importing countries. Foods 2021, 10, 425 16 of 17

Author Contributions: Conceptualization, D.W.P.; methodology, Y.S.Y.; software, Y.-U.L.; validation, D.W.P.; formal analysis, S.J.H. and H.J.K.; investigation, S.-H.K.; resources, J.P.K.; data curation, D.W.P.; writing—original draft preparation, D.W.P.; writing—review and editing, S.J.C. and H.H.K.; visualization, D.L., N.S. and Y.H.; supervision, A.G.K.; project administration, B.-S.C.; funding acquisition, J.K.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The datasets generated and/or analysed during the current study are not publicly available due data are not public but are available from the corresponding author on reasonable request. Acknowledgments: This research was supported by a research project at the Health and Environment Research Insti tute of Gwangju, South Korea. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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